Mosquitoes harbor a high diversity of RNA viruses, including many that impact human health. Despite a growing effort to describe the extent and nature of the mosquito virome, little is known about how these viruses persist, spread, and interact with both their hosts and other microbes. To address this issue we performed a metatranscriptomics analysis of 12 Western Australian mosquito populations structured by species and geographic location. Our results identified the complete genomes of 24 species of RNA viruses from a diverse range of viral families and orders, among which 19 are newly described. Comparisons of viromes revealed a striking difference between the two mosquito genera, with viromes of mosquitoes of the Aedes genus exhibiting substantially less diversity and lower abundances than those of mosquitoes of the Culex genus, within which the viral abundance reached 16.87% of the total non-rRNA. In addition, there was little overlap in viral diversity between the two genera, although the viromes were very similar among the three Culex species studied, suggesting that the host taxon plays a major role in structuring virus diversity. In contrast, we found no evidence that geographic location played a major role in shaping RNA virus diversity, and several viruses discovered here exhibited high similarity (95 to 98% nucleotide identity) to those from Indonesia and China. Finally, using abundance-level and phylogenetic relationships, we were able to distinguish potential mosquito viruses from those present in coinfecting bacteria, fungi, and protists. In sum, our metatranscriptomics approach provides important insights into the ecology of mosquito RNA viruses. IMPORTANCE Studies of virus ecology have generally focused on individual viral species. However, recent advances in bulk RNA sequencing make it possible to utilize metatranscriptomic approaches to reveal both complete virus diversity and the relative abundance of these viruses. We used such a metatranscriptomic approach to determine key aspects of the ecology of mosquito viruses in Western Australia. Our results show that RNA viruses are some of the most important components of the mosquito transcriptome, and we identified 19 new virus species from a diverse set of virus families. A key result was that host genetic background plays a more important role in shaping virus diversity than sampling location, with Culex species harboring more viruses at higher abundance than those from Aedes mosquitoes.KEYWORDS Australia, ecology, evolution, mosquito, phylogeny, transcriptomics, virome

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Ross River virus (RRV), an alphavirus of the Togaviridae family, is the most medically significant mosquito-borne virus of Australia. Past RRV phylogenetic and evolutionary analyses have been based on partial genome analyses only. Three geographically distinct RRV lineages, the Eastern, the Western, and the supposedly extinct North-Eastern lineage, were classified previously. We sought to expand on past phylogenies through robust genome-scale phylogeny to better understand RRV genetic diversity and evolutionary dynamics. We analyzed 106 RRV complete coding sequences, which included 13 genomes available on NCBI and 94 novel sequences derived for this study, sampled throughout Western Australia (1977–2014) and during the substantial Pacific Islands RRV epidemic (1979–1980). Our final data set comprised isolates sampled over 59 years (1959–2018) from a range of locations. Four distinct genotypes were defined, with the newly described genotype 4 (G4) found to be the contemporary lineage circulating in Western Australia. The prior geographical classification of RRV lineages was not supported by our findings, with evidence of geographical and temporal cocirculation of distinct genetic groups. Bayesian Markov chain Monte Carlo (MCMC) analysis revealed that RRV lineages diverged from a common ancestor approximately 94 years ago, with distinct lineages emerging roughly every 10 years over the past 50 years in periodic bursts of genetic diversity. Our study has enabled a more robust analysis of RRV evolutionary history and resolved greater genetic diversity that had been previously defined by partial E2 gene analysis. IMPORTANCE Ross River virus (RRV) causes the most common mosquito-borne infection in Australia and causes a significant burden of suffering to infected individuals as well as being a large burden to the Australian economy. The genetic diversity of RRV and its evolutionary history have so far only been studied using partial E2 gene analysis with a limited number of isolates. Robust whole-genome analysis has not yet been conducted. This study generated 94 novel near-whole-genome sequences to investigate the evolutionary history of RRV to better understand its genetic diversity through comprehensive whole-genome phylogeny. A better understanding of RRV genetic diversity will enable better diagnostics, surveillance, and potential future vaccine design.

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Detritivores are small- to medium-sized invertebrates that comminute and break down organic materials such as leaves, twigs and roots, especially within or upon the soil surface, or nearby. Detritivores constitute the majority of the invertebrate biomass pyramid in most environments and provide a key role in organic matter turnover; they also provide alternative food for polyphagous predators that can be active in pest control on crops. Many arthropod taxa are detritivores in soil and litter layers. Here, we focus on the bioindicator potential of three key detritivore groups: slaters, millipedes and oribatid mites. There are possibly 300 species of slaters (terrestrial isopods or Oniscidea) in Australia with 13 of these being introduced, mostly from north-western Europe. These non-native species are the dominant species in disturbed environments such as intensively managed forests and agricultural fields. Slaters are promising indicators of landscape disturbance, soil contamination and tillage. Millipedes are potentially important indicators of stress in agricultural landscapes, given their sensitivity to litter and soil moisture gradients and to physical and chemical perturbations. However, because there is a close association between the millipede fauna and moist plant communities in Australia, they are generally absent from drier landscapes and, therefore, their use as bioindicators in agricultural environments here is problematic. An exception to this association is the increasingly ubiquitous introduced Black Portuguese millipede. This species is tolerant of much drier conditions than most natives, and is likely to change the nature of nutrient cycling processes in pastures and native grasslands in much of southern Australia. Oribatid mites are present in all Australian terrestrial ecosystems. The few studies that have examined their response to disturbance and land use in Australia are consistent with the body of work conducted outside Australia. This consistent response means that the oribatids may be developed as indicators in agricultural, pasture and forested environments. However, the paucity of information on oribatids over appropriate spatial scales in Australia makes the use of this group extremely difficult at this time

On average, more than 1,000 individuals will acquire a mosquito-borne disease in Western Australia (WA) each year. Knowledge, attitudes, and practices (KAP) in relation to mosquitoes and mosquito-borne disease have not yet been investigated within Australia. A randomized telephone survey of 2,500 households across 12 regions in WA was undertaken between February and May 2014. The aim of the survey was to obtain baseline KAP data surrounding mosquitoes and mosquito-borne diseases in different regions of WA, across a range of age groups and between males and females. The results of this survey indicate that the majority of respondents are aware of the potential for mosquitoes in WA to transmit Ross River virus, while awareness of other endemic mosquito-borne diseases remains limited. Common misconceptions exist in relation to exotic mosquito-borne diseases, with respondents incorrectly identifying malaria and dengue as endemic diseases in WA. The survey also highlighted a range of important issues, such as limited awareness of the potential for backyard breeding in domestic containers, occupational exposure to mosquitoes in regions with a large employment base in the mining and resources sector, increased exposure to mosquitoes as a result of participation in outdoor recreational activities in the north of the State, and reduced awareness of mosquito-borne disease in individuals aged 18–34 years. The results of this study will be used to inform the development of a new communication strategy by the Department of Health, to further raise awareness of mosquito-borne disease in WA. The data will then provide a baseline against which to compare future survey results, facilitating the rigorous evaluation of new communication efforts.

Barmah Forest virus (BFV) is a medically important mosquito-borne alphavirus endemic to Australia. Symptomatic disease can be a major cause of morbidity, associated with fever, rash, and debilitating arthralgia. BFV disease is similar to that caused by Ross River virus (RRV), the other major Australian alphavirus. Currently, just four BFV whole-genome sequences are available with no genome-scale phylogeny in existence to robustly characterise genetic diversity. Thirty novel genome sequences were derived for this study, for a final 34-taxon dataset sampled over a 44 year period. Three distinct BFV genotypes were characterised (G1–3) that have circulated in Australia and Papua New Guinea (PNG). Evidence of spatio-temporal co-circulation of G2 and G3 within regions of Australia was noted, including in the South West region of Western Australia (WA) during the first reported disease outbreaks in the state’s history. Compared with RRV, the BFV population appeared more stable with less frequent emergence of novel lineages. Preliminary in vitro assessment of RRV and BFV replication kinetics found that RRV replicates at a significantly faster rate and to a higher, more persistent titre compared with BFV, perhaps indicating mosquitoes may be infectious with RRV for longer than with BFV. This investigation resolved a greater diversity of BFV, and a greater understanding of the evolutionary dynamics and history was attained.

Metagenomics revealed an impressive breadth of previously unrecognized viruses. Here, we report the virome of the Culex annulirostris Skuse mosquito, an important vector of pathogenic arboviruses in Australia. Mosquitoes were collected from three sites in the Kimberley region of Western Australia. Unbiased high-throughput sequencing (HTS) revealed the presence of 16 novel viral sequences that share less than 90% identity with known viruses. None were closely related to pathogenic arboviruses. Viruses were distributed unevenly across sites, indicating a heterogeneous Cx. annulirostris virome. Polymerase chain reaction assays confirmed HTS data and identified marked variation between the virus prevalence identified at each site.